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Non-Classical androgen actions in Sertoli cell membrane. Eloísa da Silveira Loss Department of Physiology ICBS – Federal University of Rio Grande do Sul Porto Alegre RS - Brazil. Leydig. Seminiferous tubules. Peritubular. Sertoli. Germinal. Interstitium. Testis. - PowerPoint PPT Presentation
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Non-Classical androgen actions
in Sertoli cell membrane
Eloísa da Silveira Loss
Department of Physiology
ICBS – Federal University of Rio Grande do Sul
Porto Alegre RS - Brazil
TESTIS Seminiferous epithelium
Seminiferous tubules
Testis Interstitium
Leydig
Peritubular
Sertoli
Germinal
-The interaction among the different cells of this tissue is a very dynamic and an extremely elaborate process. -Germ cells, Sertoli cells, peritubular, and Leydig cells have an elaborate network for cell-cell communication that occurs via hormones, autocrine or paracrine factors, and signalling molecules. This allows the Sertoli cells, as nursing cells, to provide developing germ cells with the required nutrients and biological factors. -Sertoli cells also present gap junctions, and these junctions provide communications alongside the seminiferous ephitelium. It occurs between Sertoli-Sertoli cells as well as between Sertoli-germ cell, giving the necessary support for the spermatogenic wave process.
Immature Sertoli cells Rat testes
5 day-old
20 day-old
Adult
Maturational development:
Sertoli cells are in proliferative and growth process.
The maturational progression is regulated by several hormones and growth factors.
Follicle-stimulating hormone (FSH) and testosterone are the two major endocrine signals that act in the testis to regulate development and spermatogenisis efficiency in adult.
Androgenic actions Classical X Non-classical
Classical Intracellular Androgen Receptor
(iAR) or nuclear androgen receptor
Non-classical Membrane Receptor
Effects only seconds or minutes after the steroid application
Androgen exert non-classical actions in a variety of cell types
This receptor have not been identified.
Probable involvement of G protein receptor and corresponding downstream signaling.
Such effects take a time lag of hours or even days
Sertoli cells membrane potential recording
Loss ES, Jacobus AP, Wassermann GF. Rapid signaling responses in Sertoli cell membranes induced by follicle stimulating hormone and testosterone: calcium inflow and electrophysiological changes. Life Sci 2011;89:577–83.
Perfusion chamber
Register electrode
Microelectrode borosilicate pipets was filled with KCl (3mM) and had a tip resistance of 15 to 25 MWThis resistance is appropriate for the
preferential impalement of cells with a size similar to that of Sertoli cells. This tip diameter makes it difficult to impale smaller cells, such as peritubular myoid cells.
1 ml with flow 1 ml/sec
Electrophysiological method Standard single microelectrode recording
Application of hormones or drugs
Intracellular recording was amplified using an Intra 767 WPI amplifier .
Grass Stimulator - Square current pulses of 0.5 nA, 0.5 Hz and 250 ms duration, applied through the intracellular electrode to estimate membrane input resistance (R0).
Oscilloscope Tektronix and its softwere (Wavestar Lite Version 1.0.10)
Standar single microelectrode recording
Register recording
Membrane potencial of Sertoli cells
Resting potential of Sertoli cells in seminiferous tubules from 12- to 15-day-old rats is very stable.The resting membrane potential average was -44±0.5 mV (n=124).Membrane resistance average was 9.3 ± 0.7 MΩ (n=124).
To avoid working with germ cells, only cells with membrane potential lower than -35 mV are included in the experiments, because this membrane potential is commonly found in Sertoli cells from normal or Sertoli-cell-enriched seminiferous tubules from immature rats.
FSH action on membrane of Sertoli cells
Wassermann, G.F., Monti Bloch L, Grillo ML, Silva FR, Loss ES, McConnell LL. Electrophysiological changes of Sertoli cells produced by the acute administration of amino acid and FSH. Hormone and metabolic research, 1992, 24(7), pp.326–8.
Testosterone produces depolarization on membrane potential of Sertoli cells
Testosterone effect at different doses on membrane potential and on membrane resistance of Sertoli cells.
Testosterone (1 µM) increases 45Ca2+ uptake within 5 min of incubation in whole rat testes (n=5)
RP 0 30 300 360 480
10M
5M
1M
0.5M
0.25M
0.1M
KRb washT
time sec
5mV
30sec
m
V
00.1M 0.25M 0.5M 1M 10M
1
2
3
0
a
a
control(ethanol)
doses of testosterone
M
oh
ms
(6)
(7)(5)
(5)
(6)
(14)
Von Ledebur, EICF, Almeida JP, Loss ES, Wassermann GF. Rapid effect of testosterone on rat Sertoli cell membrane potential. Relationship with K+ATP channels. Hormone and metabolic research, 2002, 34(10): 550–5.
Pancreatic beta-cell
-operated
VOCC
ATP sensitive K+ channels (K+ATP)
Sulphonylurea receptor (SUR) subunits generate the regulatory subunit. Inward rectifier K+ channel Kir6 subunits generate the channel pore.
K+ATP channel inhibitors glibenclamide and tolbutamide mimicked testosterone action on membrane of Sertoli cells
0 150 30030-50
-45
-40
-35
Testosterone (0.25 M)
Glibenclamide (10 M)
Resting
Tolbutamide (10 M)
**
*
Time (seconds)
Me
mb
ran
e P
ote
nti
al
(mV
)
R Te Gl To0
10
20
30resting
Testosterone (0.25 M)
Glibenclamide (10 M)
olbutamide (10M)
*
*
*
Me
mb
ran
eR
es
ista
nc
e (
M
)
Von Ledebur, EICF, Almeida JP, Loss ES, Wassermann GF. Rapid effect of testosterone on rat Sertoli cell membrane potential. Relationship with K+ATP channels. Hormone and metabolic research, 2002, 34(10), pp.550–5.
Effect of diazoxide, a K+ATP channel opener, on the
testosterone actions
-5.0
-2.5
0.0
2.5
5.0
resting potential diazoxide 400M
testosterone 10M diazoxide +testosterone
a
b
m
V (174)
(5)
(5)
(5)
C D T T+D G G+D0
500
1000
Control
Diazoxide (100 M)
Testosterone (10 M)
Testosterone + Diazoxide
Glibenclamide (20 M)
Glibenclamide + Diazoxide
* *
45C
a2+ u
pta
ke
(pm
ole
s o
f45
Ca2+
/g o
fte
ste
s)
Von Ledebur, E.I.C.F. et al., 2002. Rapid effect of testosterone on rat Sertoli cell membrane potential. Relationship with K+ATP channels. Hormone and metabolic research, 34(10), pp.550–5.
0 30 300-60
-50
-40
-30Testosterone
U73122 +Testosterone
Resting
*
*
T
TU5 min
Time (seconds)
Me
mb
ran
e P
ote
nti
al(m
V)
C T U U+T0
500
1000
Control
Testosterone
U73122 (2 M)
U73122 +Testosterone
*
45C
a2+
up
take
(pm
ole
s o
f45
Ca
2+/g
of
test
es)
Phospholipase C (PLC) inhibitor U73122 and G-protein inhibitor pertussis toxin block testosterone actions on membrane of Sertoli cells
C T PT PT+T0
200
400
600
800
Control
Testosterone (10 M)
Pertussis Toxin (1 g/ml)
Pertussis toxin + Testosterone
*
45C
a2+ u
pta
ke
(pm
ole
s o
f45
Ca2+
/g o
fte
ste
s)Loss, E.S. et al., 2004. Testosterone modulates K(+)ATP channels in Sertoli cell membrane via the PLC-PIP2 pathway. Hormone and metabolic research, 36(8), pp.519–25.
Calcium channel (VOCC) blockers: verapamil, nifedipine or Ni2+ parcialy block testosterone action on membrane potential and on the 45Ca2+ uptake.
Testosterone increase 45Ca2+ uptake in 5 minutes of incubation.
0 150 30030-50
-40
-30
-20
Testosterone
Testo+Verapamil
Testo+NifedipineTesto +Ni2+
Resting
*
Time (seconds)
Me
mb
ran
e P
ote
nti
al
(mV
)
C T T+V G G+V0
500
1000
Control
Testosterone (10 M)
Testosterone + verapamil
Glibenclamide (20 M)
*
Glibenclamide + verapamil
*
45C
a2
+ u
pta
ke
(pm
ole
s o
f4
5C
a2
+/g
of
tes
tes
)
Effect of Ca2+ channels blockers on the testosterone action
Loss, E.S. et al., 2004. Testosterone modulates K(+)ATP channels in Sertoli cell membrane via the PLC-PIP2 pathway. Hormone and metabolic research, 36(8), pp.519–25.
R?
Testosterone
β ϒGq PLC
IP3-- -- -
PIP2-- -- - DAG
K+ATP channel
ATP
K+
Ca2
+
Membrane Androgen Receptor
Pertussis toxin (PTX)
U 73122
Diazoxide
Verapamil
open
GlibenclimideTolbutamide
VOCC
Action of catechin and nandrolone at different concentrations on membrane potential
Cavalari, F.C. et al., 2012. Non-classic androgen actions in Sertoli cell membrane in whole seminiferous tubules: effects of nandrolone decanoate and catechin. Steroids, 77(1-2), pp.118–25.
The depolarizing effect of nandrolone, catechin and testosterone did not change on the presence of flutamide
Cavalari, F.C. et al., 2012. Non-classic androgen actions in Sertoli cell membrane in whole seminiferous tubules: effects of nandrolone decanoate and catechin. Steroids, 77(1-2), pp.118–25.
The depolarizing effects of nandrolone and catechin were blocked by Diazoxide, a K+
ATP channel opener, and by U73122 (2μM), an inhibitor of PLC, both 10 minutes before the topical application of the steroid and the flavonol
Cavalari, F.C. et al., 2012. Non-classic androgen actions in Sertoli cell membrane in whole seminiferous tubules: effects of nandrolone decanoate and catechin. Steroids, 77(1-2), pp.118–25.
Epitestosterone (17α-hydroxy-4-androsten-3-one)
- Epitestosterone is the 17α-epimer of testosterone.
- It has been found at similar level as testosterone in human biological fluids.
- This steroid has thus been used as a natural internal standard for assessing testosterone abuse in sports.
- It was found to possess antiandrogenic activity as well as neuroprotective effects.
Testosterone Epitestosterone
Bellemara, V et al. 2005. Characterization of 17α-hydroxysteroid dehydrogenase activity (17α-HSD) and its involvement in the biosynthesis of epitestosterone. BMC Biochemistry, 6:12.
Testosterone x Epitestosterone synthesis
Epitestosterone effect on membrane potential of Sertoli cells is similar to the testosterone effect.
De Castro, A. et al. 2013. Epitestosterone and Testosterone have similar nonclassical actions on membrane of Sertoli cells in whole seminiferous tubules. Hormone and Metabolic Research, 45: 15-21.
Photomicrographs of transverse sections of seminiferous tubules comparing the iAR-positive staining pattern on pnd 3 (A), pnd 4 (B) and adult (C) Wistar rats.
In adult animals (C), iAR was immunolocalized to peritubular cells (arrowheads), interstitial Leydig cells (arrows) and tubular Sertoli cells (asterisks). In seminiferous tubules of pups on pnd 3 (A) and pnd 4 (B), no immunostaining was detected in Sertoli cells (asterisks), whereas staining was still observed in peritubular cells (arrowheads) and interstitial Leydig cells (arrows). Some primordial germ cells are indicated by GC. Bars = 30 m.
Testosterone and epitestosterone effect on calcium uptake in whole testis from neonate rats and testosterone effect on membrane potential of Sertoli cells from rats at 5thpostnatal day
da Rosa LA, Escott GM, Cavalari FC, Schneider CMM, de Fraga LS , Loss ES. Non-classical effects ofandrogens on testes from neonatal rats. Steroids, 2014 (in press)
Crosstalk between electrophysiological actions of testosterone, epitestosterone and FSH.
da Rosa LA, Escott GM, Cavalari FC, Schneider CMM, de Fraga LS , Loss ES. Non-classical effects ofandrogens on testes from neonatal rats. Steroids, 2014 (in press)
Crosstalking effects of testosterone/ epitestosterone and FSH
CONCLUSIONS
The non-classical effect of anodrogens occurs in the membrane of Sertoli cells
This non-classical effect is through a different receptor than the iAR,
The relative importance of this receptor must be considered and further evaluated.
LABENEX